I. Adeno-Associated Virus (AAV)
Adeno-Associated Virus (AAV) is a small, naturally-occurring, non-pathogenic virus belonging to the Dependovirus genus of the Parvoviridae (Balakrishnan, B. et al. (2014) “Basic Biology of Adeno Associated Virus (AAV) Vectors Used in Gene Therapy,” Curr. Gene Ther. 14(2):86-100; Zinn, E. et al. (2014) “Adeno-Associated Virus: Fit To Serve,” Curr. Opin. Virol. 0:90-97). Despite not causing disease, AAV is known to be able to infect humans and other primates and is prevalent in human populations (Johnson, F. B. et al. (1972) “Immunological Reactivity of Antisera Prepared Against the Sodium Dodecyl Sulfate-Treated Structural Polypeptides of Adenovirus-Associated Virus,” J. Virol. 9(6):1017-1026). AAV infect a broad range of different cell types (e.g., cells of the central nervous system, heart, kidney, liver, lung, pancreas, retinal pigment epithelium or photoreceptor cells, or skeletal muscle cells). Twelve serotypes of the virus (e.g., AAV2, AAV5, AAV6, etc.), exhibiting different tissue infection capabilities (“tropisms”), have been identified (Colella, P. et al. (2018) “Emerging Issues in AAV-Mediated In Vivo Gene Therapy,” Molec. Ther. Meth. Clin. Develop. 8:87-104; Hocquemiller, M. et al. (2016) “Adeno-Associated Virus-Based Gene Therapy for CNS Diseases,” Hum. Gene Ther. 27(7):478-496; Lisowski, L. et al. (2015) “Adeno-Associated Virus Serotypes For Gene Therapeutics,” 24:59-67).
AAV is a single-stranded DNA virus that is composed of approximately 4,800 nucleotides. The viral genome may be described as having a 5′ half and a 3′ half which together comprise the genes that encode the virus' proteins (FIG. 1). The 5′ half of the AAV genome comprises the AAV rep gene, which, through the use of multiple reading frames, staggered initiating promoters (P5, P19 and P40) and alternate splicing, encodes four non-structural Rep proteins (Rep40, Rep52, Rep68 and Rep78) that are required for viral transcription control and replication and for the packaging of viral genomes into the viral capsule (Lackner, D. F. et al. (2002) “Studies of the Mechanism of Transactivation of the Adeno-Associated Virus p19 Promoter by Rep Protein,” J. Virol. 76(16):8225-8235). In the presence of viral proteins (such as Ad proteins), the P5 promoter becomes activated and mediates the transcription of the Rep68 and Rep78 proteins, which are involved in transcriptional control, in latency, in rescue, and in viral DNA replication and thus function as master controllers of the AAV life cycle (Murphy, M. et al. (2007) “Adeno-Associated Virus Type 2 p5 Promoter: a Rep-Regulated DNA Switch Element Functioning in Transcription, Replication, and Site-Specific Integration,” J. Virol. 81(8):3721-3730). Expression of the Rep68 and Rep78 proteins activates the P19 promoter, which is responsible for the transcription of the Rep40 and Rep52 proteins (Lackner, D. F. et al. (2002) “Studies of the Mechanism of Transactivation of the Adeno-Associated Virus p19 Promoter by Rep Protein,” J. Virol. 76(16):8225-8235; Ogasawara, Y. et al. (1998) “The Use of Heterologous Promoters for Adeno Associated Virus (AAV) Protein Expression in AAV Vector Production,” Microbiol. Immunol. 42(3):177-185). The 3′ half the AAV genome comprises the AAV capsid gene (cap), which encodes three capsid proteins (VP): VP1, VP2 and VP3. The three capsid proteins are translated from a single mRNA transcript that is controlled by a single promoter (P40 in case of AAV2). The 3′ half of the AAV genome also comprises the AAP gene, which encodes the AAV assembly-activating protein (AAP). Sixty VP monomers (comprising approximately 5 copies of VP1, 5 copies of VP2, and 50 copies of VP3) self-assemble around the AAV genome to form the icosahedral protein shell (capsid) of the mature viral particle (Büning, H. et al. (2019) “Capsid Modifications for Targeting and Improving the Efficacy of AAV Vectors,” Mol. Ther. Meth. Clin. Devel. 12:P248-P265; Van Vliet K. M. et al. (2008) The Role of the Adeno-Associated Virus Capsid in Gene Transfer. In: DRUG DELIVERY SYSTEMS, Jain, K. K. (eds.), Meth. Molec. Biol. 437:51-91). The AAV AAP protein is believed to be required for stabilizing and transporting newly produced VP proteins from the cytoplasm into the cell nucleus. The 3′ half of the AAV genome also comprises the AAV X gene, which is believed to encode a protein that supports genome replication (Colella, P. et al. (2018) “Emerging Issues in AAV-Mediated In Vivo Gene Therapy,” Molec. Ther. Meth. Clin. Develop. 8:87-104; Büning, H. et al. (2019) “Capsid Modifications for Targeting and Improving the Efficacy of AAV Vectors,” Mol. Ther. Meth. Clin. Devel. 12:P248-P265; Cao, M. et al. (2014) “The X Gene Of Adeno-Associated Virus 2 (AAV2) Is Involved In Viral DNA Replication,” PLoS ONE 9, e104596:1-10).
The above-described AAV gene-coding sequences are flanked by two AAV-specific palindromic inverted terminal repeated sequences (ITR) of 145 nucleotides (Balakrishnan, B. et al. (2014) “Basic Biology of Adeno-Associated Virus (AAV) Vectors Used in Gene Therapy,” Curr. Gene Ther. 14(2):86-100; Colella, P. et al. (2018) “Emerging Issues in AAV-Mediated In Vivo Gene Therapy,” Molec. Ther. Meth. Clin. Develop. 8:87-104).
AAV is an inherently defective virus, lacking the capacity to perform at least two critical functions: the ability to initiate the synthesis of viral-specific products and the ability to assemble such products to form the icosahedral protein shell (capsid) of the mature infectious viral particle. It thus requires a co-infecting “helper” virus, such as adenovirus (Ad), herpes simplex virus (HSV), cytomegalovirus (CMV), vaccinia virus or human papillomavirus to provide the viral-associated (VA) RNA that is not encoded by the genes of the AAV genome. Such VA RNA is not translated, but plays a role in regulating the translation of other viral genes. Similarly, the AAV genome does not include genes that encode the viral proteins E1a, E1b, E2a, and E4; thus, these proteins must also be provided by a co-infecting “helper” virus. The E1a protein greatly stimulate viral gene transcription during the productive infection. The E1b protein block apoptosis in adenovirus-infected cells, and thus allow productive infection to proceed. The E2a protein plays a role in the elongation phase of viral strand displacement replication by unwinding the template and enhancing the initiation of transcription. The E4 protein has been shown to affect transgene persistence, vector toxicity and immunogenicity (see, Grieger, J. C. et al. (2012) “Adeno-Associated Virus Vectorology, Manufacturing, and Clinical Applications,” Meth. Enzymol. 507:229-254; Dyson, N. et al. (1992) “Adenovirus E1A Targets Key Regulators Of Cell Proliferation,” Canc. Surv. 12:161-195; Jones N. C. (1990) “Transformation By The Human Adenoviruses,” Semin. Cancer Biol. 1(6):425-435; Ben-Israel, H. et al. (2002) “Adenovirus and Cell Cycle Control,” Front. Biosci. 7:d1369-d1395; Hoeben, R. C. et al. (2013) “Adenovirus DNA Replication,” Cold Spring Harb. Perspect. Biol. 5:a013003 (pages 1-11); Berk, A. J. (2013) “Adenoviridae: The Viruses And Their Replication, In: FIELDS VIROLOGY, 6th Edition (Knipe, D. M. et al., Eds.), Vol. 2., Lippincott Williams & Wilkins, Philadelphia, pages 1704-1731; Weitzman, M. D. (2005) “Functions Of The Adenovirus E4 Proteins And Their Impact On Viral Vectors,” Front. Biosci. 10:1106-1117).
AAV viruses infect both dividing and non-dividing cells, and persist as circular episomal molecules or can be integrated into the DNA of a host cell at specific chromosomic loci (Adeno-Associated Virus Integration Sites or AAV5) (Duan, D. (2016) “Systemic Delivery Of Adeno-Associated Viral Vectors,” Curr. Opin. Virol. 21:16-25; Grieger, J. C. et al. (2012) “Adeno-Associated Virus Vectorology, Manufacturing, and Clinical Applications,” Meth. Enzymol. 507:229-254). AAV remains latent in such infected cells unless a helper virus is present to provide the functions needed for AAV replication and maturation.
II. rAAV and their Use in Gene Therapy
In light of AAV's properties, recombinantly-modified versions of AAV (rAAV) have found substantial utility as vectors for gene therapy (see, Naso, M. F. et al. (2017) “Adeno-Associated Virus (AAV) as a Vector for Gene Therapy,” BioDrugs 31:317-334; Berns, K. I. et al. (2017) “AAV: An Overview of Unanswered Questions,” Human Gene Ther. 28(4):308-313; Berry, G. E. et al. (2016) “Cellular Transduction Mechanisms Of Adeno-Associated Viral Vectors,” Curr. Opin. Virol. 21:54-60; Blessing, D. et al. (2016) “Adeno-Associated Virus And Lentivirus Vectors: A Refined Toolkit For The Central Nervous System,” 21:61-66; Santiago-Ortiz, J. L. (2016) “Adeno-Associated Virus (AAV) Vectors in Cancer Gene Therapy,” J. Control Release 240:287-301; Salganik, M. et al. (2015) “Adeno-Associated Virus As A Mammalian DNA Vector,” Microbiol. Spectr. 3(4):1-32; Hocquemiller, M. et al. (2016) “Adeno-Associated Virus-Based Gene Therapy for CNS Diseases,” Hum. Gene Ther. 27(7):478-496; Lykken, E. A. et al. (2018) “Recent Progress And Considerations For AAV Gene Therapies Targeting The Central Nervous System,” J. Neurodevelop. Dis. 10:16:1-10; Büning, H. et al. (2019) “Capsid Modifications for Targeting and Improving the Efficacy of AAV Vectors,” Mol. Ther. Meth. Clin. Devel. 12:P248-P265; During, M. J. et al. (1998) “In Vivo Expression Of Therapeutic Human Genes For Dopamine Production In The Caudates Of MPTP-Treated Monkeys Using An AAV Vector,” Gene The. 5:820-827; Grieger, J. C. et al. (2012) “Adeno-Associated Virus Vectorology, Manufacturing, and Clinical Applications,” Meth. Enzymol. 507:229-254; Kotterman, M. A. et al. (2014) “Engineering Adeno-Associated Viruses For Clinical Gene Therapy,” Nat. Rev. Genet. 15(7):445-451; Kwon, I. et al. (2007) “Designer Gene Delivery Vectors: Molecular Engineering and Evolution of Adeno-Associated Viral Vectors for Enhanced Gene Transfer,” Pharm. Res. 25(3):489-499; U.S. Pat. Nos. 10,266,845; 10,081,659; 9,890,396; 9,840,719; 9,839,696; 9,834,789; 9,803,218; 9,783,825; 9,777,291; 9,540,659; 9,527,904; 8,236,557; 7,972,593 and 7,943,374).
rAAV are typically produced using circular plasmids (“rAAV plasmid vector”). The AAV rep and cap genes are typically deleted from such constructs and replaced with a promoter, a β-globin intron, a cloning site into which a therapeutic gene of choice (transgene) has been inserted, and a poly-adenylation (“polyA”) site. The inverted terminal repeated sequences (ITR) of the rAAV are, however, retained, so that the transgene expression cassette of the rAAV plasmid vector is flanked by AAV ITR sequences (Colella, P. et al. (2018) “Emerging Issues in AAV-Mediated In Vivo Gene Therapy,” Molec. Ther. Meth. Clin. Develop. 8:87-104; Büning, H. et al. (2019) “Capsid Modifications for Targeting and Improving the Efficacy of AAV Vectors,” Mol. Ther. Meth. Clin. Devel. 12:P248-P265). Thus, in the 5′ to 3′ direction, the rAAV comprises a 5′ ITR, the transgene expression cassette of the rAAV, and a 3′ ITR.
rAAV have been used to deliver a transgene to patients suffering from any of a multitude of genetic diseases (e.g., hereditary lipoprotein lipase deficiency (LPLD), Leber's congenital amaurosis (LCA), aromatic L-amino acid decarboxylase deficiency (AADC), choroideremia and hemophilia), and have utility in new clinical modalities, such as in interfering RNA (RNAi) therapy and gene-modifying strategies such as Crispr/Cas9 (U.S. Pat. Nos. 8,697,359, 10,000,772, 10,113,167, 10,227,611; Lino, C. A. et al. (2018) “Delivering CRISPR: A Review Of The Challenges And Approaches,” Drug Deliv. 25(1):1234-1237; Ferreira, V. et al. (2014) “Immune Responses To AAV-Vectors, The Glybera Example From Bench To Bedside” Front. Immunol. 5(82):1-15), Büning, H. et al. (2019) “Capsid Modifications for Targeting and Improving the Efficacy of AAV Vectors,” Mol. Ther. Meth. Clin. Devel. 12:P248-P265; Rastall, D. P. W. (2017) “Current and Future Treatments for Lysosomal Storage Disorders,” Curr. Treat Options Neurol. 19(12):45; Kay, M. et al. (2017) “Future Of rAAV Gene Therapy: Platform For RNAi, Gene Editing And Beyond,” Human Gene Ther. 28:361-372); Berns, K. I. et al. (2017) “AAV: An Overview of Unanswered Questions,” Human Gene Ther. 28(4):308-313). More than 150 clinical trials involving rAAV have been instituted (Büning, H. et al. (2019) “Capsid Modifications for Targeting and Improving the Efficacy of AAV Vectors,” Mol. Ther. Meth. Clin. Devel. 12:P248-P265; Clément, N. et al. (2016) “Manufacturing Of Recombinant Adeno Associated Viral Vectors For Clinical Trials,” Meth. Clin. Develop. 3:16002:1-7). The most commonly used AAV serotype for such recombinantly-modified AAV is AAV2, which is capable of infecting cells of the central nervous system, kidney, retinal pigment epithelium and photoreceptor cells. AAV serotype is AAV9, which infects muscle cells, also has been widely used (Duan, D. (2016) “Systemic Delivery Of Adeno-Associated Viral Vectors,” Curr. Opin. Virol. 21:16-25). AAV serotypes are described in U.S. Pat. Nos. 10,301,650; 10,266,846; 10,265,417; 10,214,785; 10,214,566; 10,202,657; 10,046,016; 9,884,071; 9,856,539; 9,737,618; 9,677,089; 9,458,517; 9,457,103; 9,441,244; 9,193,956; 8,846,389; 8,507,267; 7,906,111; 7,479,554; 7,186,552; 7,105,345; 6,984,517; 6,962,815; and 6,733,757.
III. Methods of rAAV Production
rAAV containing a desired transgene expression cassette are typically produced by human cells (such as HEK293) grown in suspension. Since, as described above, rAAV are defective viruses, additional functions must be provided in order to replicate and package rAAV.
rAAV can be produced by transiently transfecting cells with an rAAV plasmid vector and a second plasmid vector that comprises an AAV helper function-providing polynucleotide that provides the Rep52 and Rep78 genes that are required for vector transcription control and replication, and for the packaging of viral genomes into the viral capsule (Rep40 and Rep68 are not required for rAAV production) and the cap genes that were excised from the AAV in order to produce the rAAV. The second plasmid vector may additionally comprise a non-AAV helper function-providing polynucleotide that encodes the viral transcription and translation factors (E1a, E1b, E2a, VA and E4) required for AAV proliferation, so as to comprise, in concert with the rAAV, a double plasmid transfection system (Grimm, D. et al. (1998) “Novel Tools For Production And Purification Of Recombinant Adeno-Associated Virus Vectors,” Hum. Gene Ther. 9:2745-2760; Penaud-Budloo, M. et al. (2018) “Pharmacology of Recombinant Adeno-associated Virus Production,” Molec. Ther. Meth. Clin. Develop. 8:166-180).
However, it has become increasingly common to clone the AAV helper function-providing polynucleotide (which provides the required rep and cap genes) into an AAV helper plasmid, and to clone the non-AAV helper function-providing polynucleotide (which provides the genes that encode the viral transcription and translation factors) on a different plasmid (e.g., an “Ad helper plasmid”), so that such plasmids, in concert with an rAAV plasmid vector, comprise a triple plasmid transfection system (FIG. 2). Use of the triple plasmid transfection system has the advantage of permitting one to easily switch one cap gene for another, thereby facilitating changes in the rAAV's serotype. The use of helper plasmids, rather than helper viruses, permits rAAV to be produced without additionally producing particles of the helper virus (François, A. et al. (2018) “Accurate Titration of Infectious AAV Particles Requires Measurement of Biologically Active Vector Genomes and Suitable Controls,” Molec. Ther. Meth. Clin. Develop. 10:223-236; Matsushita, T. et al. (1998) “Adeno-Associated Virus Vectors Can Be Efficiently Produced Without Helper Virus,” Gene Ther. 5:938-945).
The transient transfection of plasmid DNAs comprising the rAAV plasmid vector, the AAV rep and cap genes, and the trans-acting AAD helper genes into HEK293 cells by calcium phosphate coprecipitation has become the standard method to produce rAAV in the research laboratory (Grimm, D. et al. (1998) “Novel Tools For Production And Purification Of Recombinant Adeno-Associated Virus Vectors,” Hum. Gene Ther. 9:2745-2760). However, the use of such a calcium phosphate-mediated transfection process with suspension-cultured transfected mammalian cells requires media exchanges, and is thus not considered ideal for the large-scale rAAV production that is required in order to produce therapeutic doses of rAAV (Lock, M. et al. (2010) “Rapid, Simple, and Versatile Manufacturing of Recombinant Adeno-Associated Viral Vectors at Scale,” Hum. Gene Ther. 21:1259-1271). For this reason, polyethylenimine (PEI), has been used as a transfection reagent and has been found to provide yields of virus that are similar to those obtained using calcium phosphate-mediated transfection (Durocher, Y. et al. (2007) “Scalable Serum-Free Production Of Recombinant Adeno-Associated Virus Type 2 By Transfection Of 293 Suspension Cells,” J. Virol. Meth. 144:32-40).
rAAV may alternatively be produced in insect cells (e.g., sf9 cells) using baculoviral vectors (see, e.g., U.S. Pat. Nos. 9,879,282; 9,879,279; 8,945,918; 8,163,543; 7,271,002 and 6,723,551), or in HSV-infected baby hamster kidney (BHK) cells (e.g., BHK21) (François, A. et al. (2018) “Accurate Titration of Infectious AAV Particles Requires Measurement of Biologically Active Vector Genomes and Suitable Controls,” Molec. Ther. Meth. Clin. Develop. 10:223-236). Methods of rAAV production are reviewed in Grieger, J. C. et al. (2012) “Adeno-Associated Virus Vectorology, Manufacturing, and Clinical Applications,” Meth. Enzymol. 507:229-254, and in Penaud-Budloo, M. et al. (2018) “Pharmacology of Recombinant Adeno-associated Virus Production,” Molec. Ther. Meth. Clin. Develop. 8:166-180.
IV. Methods of rAAV Purification and Recovery
After production, rAAV are typically collected and purified by one or more overnight CsCl gradient centrifugations (Zolotukhin, S. et al. (1999) “Recombinant Adeno Associated Virus Purification Using Novel Methods Improves Infectious Titer And Yield,” Gene Ther. 6:973-985), followed by desalting to form a purified rAAV production stock. Titers of 1012-1013 infectious rAAV capsids/mL are obtainable.
Because rAAV infection does not cause a cytopathic effect, plaque assays cannot be used to determine the infectious titer of an rAAV preparation. Infectious titer is thus typically measured as the median tissue culture infective dose (TCID50). In this method, a HeLa-derived AAV2 rep- and cap-expressing cell line is grown in a 96-well plate and infected with replicate 10-fold serial dilutions of the rAAV preparation, in the presence of adenovirus of serotype 5. After infection, vector genome replication is determined by quantitative PCR (qPCR) (Zen, Z. et al. (2004) “Infectious Titer Assay For Adeno-Associated Virus Vectors With Sensitivity Sufficient To Detect Single Infectious Events,” Hum. Gene Ther. 15:709-715). Alternatively, the infectious titer of an rAAV preparation can be measured using the infectious center assay (ICA). This assay uses HeLa rep-cap cells and Ad, but, after incubation, involves transferring the cells to a membrane. A labeled probe that is complementary to a portion of the employed transgene is used to detect infectious centers (representing individual infected cells) via hybridization. Although more widely used, the TCID50 assay has been reported to lead to a higher background than the ICA and to overestimate vector infectivity relative to the ICA (François, A. et al. (2018) “Accurate Titration of Infectious AAV Particles Requires Measurement of Biologically Active Vector Genomes and Suitable Controls,” Molec. Ther. Meth. Clin. Develop. 10:223-236). Methods of producing and purifying rAAV are described inter alia in U.S. Pat. Nos. 10,294,452; 10,161,011; 10,017,746; 9,598,703; 7,625,570; 7,439,065; 7,419,817; 7,208,315; 6,995,006; 6,989,264; 6,846,665 and 6,841,357.
Despite all such prior advances, a need remains to develop methods capable of addressing problems that presently limit the applicability of rAAV to gene therapy (Grieger, J. C. et al. (2012) “Adeno-Associated Virus Vectorology, Manufacturing, and Clinical Applications,” Meth. Enzymol. 507:229-254; Kotterman, M. A. et al. (2014) “Engineering Adeno-Associated Viruses For Clinical Gene Therapy,” Nat. Rev. Genet. 15(7):445-451; Kwon, I. et al. (2007) “Designer Gene Delivery Vectors: Molecular Engineering and Evolution of Adeno-Associated Viral Vectors for Enhanced Gene Transfer,” Pharm. Res. 25(3):489-499; Naso, M. F. et al. (2017) “Adeno-Associated Virus (AAV) as a Vector for Gene Therapy,” BioDrugs 31:317-334).
The present invention is directed to improved methods for increasing the efficiency of AAV and rAAV packaging through regulation of the expression of the AAV rep and cap genes.